Inhibition of Autotaxin and LPA Receptor

1-oleoyl-LPC (18:1) was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and diluted in Dulbecco’s Modified Eagle’s Medium (Lonza, Walkersville, MD, USA) including 0.1% bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA). The selective ATX inhibitor PAT-048^{21 (link),22 (link),24 (link)} and the LPA₁-selective small molecule antagonist AM152 (Example 1 in patent application US/20120015991) were synthesized by PharmAkea Inc (San Diego, CA, USA). Sodium pyruvate, NEAA mixture and Penicillin/Streptomycin were purchased from Lonza. L-glutamine was from CellGenix (Portsmouth, NH, USA). Dulbecco’s Modified Eagle’s Medium and fetal bovine serum (FBS) were from Thermo Fisher Scientific (Waltham, MA, USA). Charcoal-stripped FBS was prepared as previously described. In brief, 10 mL of FBS was incubated with 1 g of activated charcoal (Sigma Aldrich) overnight at 4 °C^{42 (link)}. After centrifugation at 2000 × g for ten minutes, the supernatant was filtered for the experiments. DMSO was from Sigma Aldrich. Mouse primary renal fibroblasts were obtained from Cell Biologics (Chicago, IL, USA). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. Recombinant ATX was purchased from R&D systems (Minneapolis, MN, USA).